PCR Mediated Gene Cloning
There are three strategies for cloning PCR products-
1) T/A cloning is the easiest cloning method. T/A cloning takes advantage of the terminal transferase activity of Taq polymerase and other non-proofreading DNA polymerases which adds a single 3'-A overhang to each end of the PCR product. The resulting PCR product is then ligated into a linear vector with a 3´ terminal 'T' or 'U' at both ends.
2) Directional cloning. A restriction enzyme target site is introduced into each of the PCR primers. The resulting PCR product and cloning vector are digested with the restriction enzymes to generate complementary ends at the PCR product and the vector which are then ligated.
3) Blunt-end cloning-Blunt-end PCR product generated by proof-reading polymerase such as the Pfu DNA Polymerase can also be cloned into a blunt-end vector.
The cloning of PCR-amplified fragments into a linear vector is typically a rapid and efficient process. However, not all PCR fragments will clone with the same efficiency into the same vector. These differences may be due to fragment size, insert toxicity, and the complexity of the insert. The size of the fragment being cloned is a primary contributor to the overall cloning efficiency. Large fragments of DNA (≥ 5 kb) are amenable to cloning in high-copy number vectors, yet at a much lower efficiency. Optimization of molar concentration ratios of the vector to insert is critical to ensure efficient cloning. Successful cloning ratios may range from 1:1 to 1:10. For example, if the vector is 3 kb and the insert is 1 kb, one-third the amount of insert needs to be added to attain a 1:1 molar ratio.
There are three strategies for cloning PCR products-
1) T/A cloning is the easiest cloning method. T/A cloning takes advantage of the terminal transferase activity of Taq polymerase and other non-proofreading DNA polymerases which adds a single 3'-A overhang to each end of the PCR product. The resulting PCR product is then ligated into a linear vector with a 3´ terminal 'T' or 'U' at both ends.
2) Directional cloning. A restriction enzyme target site is introduced into each of the PCR primers. The resulting PCR product and cloning vector are digested with the restriction enzymes to generate complementary ends at the PCR product and the vector which are then ligated.
3) Blunt-end cloning-Blunt-end PCR product generated by proof-reading polymerase such as the Pfu DNA Polymerase can also be cloned into a blunt-end vector.
The cloning of PCR-amplified fragments into a linear vector is typically a rapid and efficient process. However, not all PCR fragments will clone with the same efficiency into the same vector. These differences may be due to fragment size, insert toxicity, and the complexity of the insert. The size of the fragment being cloned is a primary contributor to the overall cloning efficiency. Large fragments of DNA (≥ 5 kb) are amenable to cloning in high-copy number vectors, yet at a much lower efficiency. Optimization of molar concentration ratios of the vector to insert is critical to ensure efficient cloning. Successful cloning ratios may range from 1:1 to 1:10. For example, if the vector is 3 kb and the insert is 1 kb, one-third the amount of insert needs to be added to attain a 1:1 molar ratio.
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